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Cardiovascular Research 2007 76(3):528-538; doi:10.1016/j.cardiores.2007.08.002
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Copyright © 2007, European Society of Cardiology

Endothelial lipase enhances low density lipoprotein binding and cell association in THP-1 macrophages

Guosong Qiu and John S. Hill*

Atherosclerosis Specialty Laboratory, Healthy Heart Program, St. Paul's Hospital, James Hogg iCAPTURE Centre for Cardiovascular and Pulmonary Research, University of British Columbia, Vancouver, Canada BC V6Z 1Y6

*Correspondence author. Healthy Heart Program, St. Paul's Hospital, 1081 Burrard Street, Vancouver, Canada BC V6Z 1Y6. Tel.: +1 604 806 8616; fax: +1 604 806 8590. jshill{at}interchange.ubc.ca

Received 10 April 2007; revised 18 July 2007; accepted 5 August 2007


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 
Objective Endothelial lipase (EL) is expressed in macrophages in human atherosclerotic lesions. However, its specific metabolic role in human macrophages has not been fully explored.

Methods The present study used lentivirus containing either shRNA or cDNA for EL to decrease or increase EL expression, respectively in THP-1 macrophages to investigate the consequence on LDL binding and cell association.

Results EL suppression significantly decreased the binding and cell association of native LDL (52% and 33%) and mildly oxLDL (43% and 36%) as well as extensively oxLDL binding (36%) in THP-1 macrophages. EL overexpression markedly increased the binding and cell association of native LDL (3.1- and 2.2-fold), mildly oxLDL (1.9- and 1.4-fold), and extensively oxLDL (1.5- and 1.5-fold). An inactive mutant EL compromised EL-mediated cell association of native and mildly oxLDL but not extensively oxLDL. Heparinase treatment almost completely eliminated EL-mediated native and oxLDL binding and cell association in THP-1 macrophages. LDL receptor blocking by antibodies decreased EL-mediated native LDL binding and cell association by 24% and 54%, respectively. Neither receptor associated protein or CD36 antibody treatment led to changes in EL-mediated lipoprotein binding and cell association. Furthermore, wild-type and the catalytically inactive mutant EL increased lipid accumulation in THP-1 macrophages.

Conclusions EL expression promotes the binding and uptake of native and oxidized LDL in THP-1 macrophages in a heparan sulfate proteoglycan-dependent manner, and the LDL receptor was partly responsible for the EL-enhanced uptake of native LDL.

KEYWORDS Macrophages; Lipoproteins; Lipid metabolism; Atherosclerosis; Endothelial lipase


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 References
 
Endothelial lipase (EL) is member of a family of several mammalian lipases which hydrolyse triglycerides and phospholipids and is expressed in a variety of tissues including endothelial cells and macrophages [1–3]. EL preferentially hydrolyzes phospholipids and genetic variants in humans have been shown to modulate the concentration of high density lipoprotein (HDL) [4–6]. Clinical studies have reported that the concentration of EL protein in post-heparin plasma was increased in a population of moderately obese men and correlated with the concentration of several inflammatory biomarkers [7]. In a separate cohort of 858 healthy participants with a family history of heart disease, post-heparin plasma EL protein concentration was associated with metabolic syndrome and coronary artery calcification [8]. Additional evidence that EL may have a proatherogenic role is provided in a report describing increased expression of EL in atherosclerotic samples isolated from human coronary arteries [9]. However, the results obtained from atherosclerosis studies using EL knock out mouse models are not consistent. Ishida et al. reported the EL knockout in apoE-deficient mice lessened the aortic atherosclerosis by ~70% on both regular and chow diets [10] whereas no differences were observed in a second study where both apoE-deficient and LDLR-deficient mice were utilized [11]. Animal studies have also shown that EL can modulate the plasma levels of apoB-containing lipoproteins, but to a lesser extent than HDL [2,4–6,12]. In vitro studies have shown that EL expression in transfected Chinese Hamster Ovary (CHO) cells resulted in a marked increase of LDL binding and uptake [13], an effect dependent on the presence of cell surface heparan sulfate proteoglycans (HSPGs).

There is evidence that the role of lipases in atherosclerosis is dependent on its tissue origin. For example, the systemic expression of LPL was associated with varying outcomes in animal models [14–18], whereas macrophage-specific expression of LPL consistently demonstrated a proatherogenic phenotype [19–21]. However, there have been no studies which have directly addressed the functional role of human macrophage-derived EL expression.

In the present study, we have used lentivirus transduction of THP-1 monocytes to facilitate the suppression or increased expression of endogenous EL. We have observed that EL expression is associated with the binding and cell association of native and oxidized LDL (oxLDL) in THP-1 derived macrophages and that cell surface HSPG were necessary for this effect.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 References
 
2.1 Cell culture
Human THP-1 monocytes from American Type Culture Collection were cultured in RPMI1640 medium supplemented with 10% FBS, 1 mM sodium pyruvate (Invitrogen), 1.5% sodium bicarbonate (Invitrogen), and 1% antibiotic–antimycotic solution (Invitrogen). THP-1 cells within 20 passages were differentiated into macrophages for experimental use. 293T HEK cells for lentivirus production were cultured in DMEM medium supplemented with 10% FBS and 1% antibiotic–antimyotic solution. All cells were maintained in humidified cell culture incubator at 5% CO2 and 95% air.

2.2 Lentiviral production
EL shRNA integrated into lentivirus was used to suppress EL expression (Loss of Function/LOF). The construction of shRNA-containing lentivirus has been described previously [22]. Briefly, annealed shRNA oligos were inserted into an entry vector pSHAG (kind gift from Dr. Greg Hannon, Cold Spring Harbor Laboratory) and then transferred into lentiviral vector pHR-CMV-EGFP. Lentivirus was packaged in 293T HEK cell by contransfecting pHR-CMV-eGFP and packaging vectors (pMD.G and pCMV.{theta}R8.2). Collected viral supernatants were concentrated and titrated in 293T HEK cells using FACS.

Overexpression (Gain of Function/GOF) of wild-type EL (EL-WT) was also achieved by lentivirus. Briefly, EL cDNA sequence extracted from pcDNA5-FRT-EL (kind gift from Howard Wong, UCLA) was inserted into PmeI site of lentiviral vector pWPI-eGFP (named pWPI-eGFP-EL). After the confirmation of cDNA direction and sequence fidelity by DNA sequencing, lentivirus was produced by co-transfecting pWPI-eGFP-EL with packaging vectors (pMD.2G and psPAX2).

Catalytically inactive EL (EL-S149A) was generated by site-directed mutagenesis using template plasmid pWPI-eGFP-EL, primers (sense: 5'-ACTTGATCGGCTACGCCCTCGGAGCGCACG-3'; antisense: 5'-CGTGCGCTCCGAGGGCGTAGCCGATCAAGT-3'), and QuikChange XL site-directed mutagenesis kit (Stratagene). The S149->A mutation was confirmed by DNA sequencing. The generation of lentivirus expressing EL-S149A was performed in the same way as described above.

Lentivirus were then used to transduce THP-1 monocytes (5x105 cells in 12-well plates) at multiplicity of infection (MOI) of 5, 10 and 20. After 48 h, monocytes were collected and analyzed for eGFP positivity using FACS. The eGFP expression was also assessed by fluoroscopy in macrophages differentiated from THP-1 monocytes by three-day PMA stimulation. For EL's LOF and GOF studies, lentivirus containing either scrambled shRNA or void insert served as controls, respectively.

2.3 mRNA extraction and real-time qRT-PCR
Lentivirus-transduced THP-1 monocytes were differentiated into macrophages by the stimulation of PMA at 100 nM for 3 days. Thereafter, total cellular RNA was isolated, and EL mRNA was quantitated using SuperScriptTM III One-Step RT-PCR system (Invitrogen) and Assay-On-DemandTM Gene Expression primer sets (18S and EL, Applied Biosystems) on the ABI Prism® 7900 platform at the following conditions: 50 °C for 30 min, 95 °C for 15 min, and 40 cycles of 95 °C for 15 s and 60 °C for 60 s. Serially-diluted samples from concentrated RNA were used to create standard curves.

2.4 Phospholipase activity assay
In-well phospholipase assay was used to evaluate the total phospholipase activity. Briefly, the phospholipase substrate bis-BODIPY FL C11-PC (Invitrogen) was sonicated in PBS and then added to cell culture at a final concentration of 2 µg/mL. After 4 h incubation, culture medium aliquots were collected for the measurement of fluorescent intensity at excitation and emission wavelengths of 488 nm and 530 nm, respectively. Enzyme activity was normalized for total cell protein.

2.5 Western blot
THP-1 macrophages (5x106 cells) were lysed in RIPA lysis buffer supplemented with protease inhibitor cocktail (Sigma). Total protein (20 µg) of lysates or 20 µl conditioned medium were used for SDS-PAGE electrophoresis and western blot analysis following conventional procedure and using a primary EL antibody (Cayman Chemical). Chemiluminescent signals were captured and quantitated by ChemiGenius2 image system.

2.6 DiI labeling and oxidation of LDL
Native LDL (Biomedical Technologies) was incubated with 300 µg/ml 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) at 37 °C overnight. Thereafter, both DiI-labeled and unlabeled LDLs were oxidized by 5 µM CuSO4 for 2 h or overnight at 37 °C for mildly and extensively oxidized LDL (oxLDL) preparations, respectively. After oxidation termination by 100 nM EDTA, LDL was re-isolated into PBS using a PD-10 desalting column (GE Healthcare). Thiobarbituric acid reacting substances (TBARS) assay (ZeptoMetrix) was used to evaluate the oxidation extent of LDL. A LDL preparation with a TBARS value of 20–30 nmol/mg protein of MDA equivalents was classified as mildly oxidized LDL whereas TBARS values greater than 50 nmol/mg protein of MDA equivalents was classified as extensively oxidized LDL.

2.7 LDL binding and cell association assay
THP-1 monocytes (5x105 cells) were transduced with lentivirus at MOI of 20 for 2 days, then differentiated into macrophages by 100 nM PMA for additional 2 days. Thereafter, THP-1 macrophages were incubated in 5% lipoprotein deficient serum (LPDS) RPMI 1640 medium for 24 h. Subsequently, THP-1 macrophages were replenished with 5% LPDS-RPMI 1640 medium with 10ug/ml DiI-LDL (native, mildly oxidized, and extensively oxidized LDLs), and incubated at 4 °C or 37 °C for LDL binding and cell association assays, respectively. Cell association of LDL at 37 °C represents the cell surface-bound and internalized LDL. For background binding/uptake, 20-fold excess of non-DiI-labeled LDL was added in competition with DiI-LDL. After 4 hour incubation, cells were washed twice with 0.2% BSA-PBS and PBS, and then lysed in RIPA buffer. Cell lysates were measured for fluorescence at the excitation and emission wavelengths of 520 nm and 580 nm. The cell bound or associated LDL was normalized for cellular protein.

During LDL binding and cell association assay, heparinase I (2.5 units/ml) was added into culture medium to inhibit non-catalytic function (bridging function) of EL. Moreover, LDL receptor monoclonal antibody (15 µg/ml, Fitzgerald), CD36 monocolonal antibody (10 µg/ml, Novus Biochemicals), and Receptor Associated Protein (RAP, which blocks LRP at the concentration of 4 µg/ml, Calbiochem) were added into cell culture 1 h before the addition of DiI-LDL to block corresponding lipoprotein receptors.

2.8 Foam cell evaluation by Oil Red O staining
THP-1 derived macrophages (5x105cells/well) at different treatments (LOF or GOF) were incubated in 5% LPDS-RPMI 1640 medium containing 20 µg/ml LDL of different oxidation degrees for 24 hours, thereafter, cells were washed once with PBS and fixed in 4% paraformaldehyde for 10 min. After rinse with 60% isopropanol, cells were stained with 0.3% Oil Red O in 60% isopropanol for 10 min. Next, cells were rinsed again with 60% isopropanol, counterstained by hematoxylin for 3 min. After copious wash with distilled water, cells were then photographed under a microscope at 200x magnification.

2.9 Statistical analysis
All parametric data were represented with mean±standard error of the mean, data were input into Prism 4 for Windows and analyzed using either student t test or two-way ANOVA. p value<0.05 was considered to be statistically significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 References
 
3.1 Lentiviral transduction successfully altered EL expression in THP-1 macrophages
We have previously reported that an MOI of 20 was consistent with 100% transduction efficiency for THP-1 monocytes and that a selected siRNA sequence specific for the human EL gene was able to suppress EL mRNA levels by 76%–94% with a corresponding >70% decrease in EL protein as assessed by Western blotting [22]. The transduction of THP-1 monocytes with lentivirus containing the EL cDNA markedly increased EL mRNA by >100 fold for both wild-type EL (EL-WT) and S149A mutated EL (EL-S149A) (Fig. 1A). As shown by Western blot analysis, the levels of both wild-type and mutated EL in cultured medium were dramatically increased, in comparison with non-detectable EL protein levels in control THP-1 macrophages after lentiviral transduction (Fig. 1B). By contrast, the levels of cell-associated EL protein as analyzed in the cell lysate was only increased by 4.4- and 3.8-fold for wild-type and S149A mutated EL, respectively (Fig. 1C), suggesting that the majority of mature EL protein was secreted into the extracellular compartment. Total phospholipase activity was elevated by 50% after transduction with wild-type EL cDNA whereas transduction of the S149A mutant EL did not cause an appreciable increase in total phospholipase activity (Fig. 1A).


Figure 1
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  		Fig. 1 EL overexpression by lentiviral transduction in THP-1 derived macrophages. THP-1 monocytese (5x105 cells/well) were transduced with lentivirus (Control: no cDNA insert; EL-WT: lentivirus containing wild-type EL; EL-S149A: lentivirus containing S149->A mutated EL) at a MOI of 20 for two days before differentiated into macrophages by 3-day 100 nM PMA stimulation. Total RNA was analyzed for EL expression by real-time qRT-PCR. Total in-well phospholipase activity was used to detect the phospholipase activity in heparin-challenged culture medium. Cell lysate was collected from macrophages without heparin challenge and conditioned media were analyzed by Western blot for EL protein. Panel A: EL mRNA level after lentiviral transduction (***p<0.001, n=4 from two separate experiments) and total phospholipase activity (***p<0.001, n=4 from two separate experiments); EL protein was also probed by Western blot in conditioned medium (Panel B) and cell lysate (Panel C) (a representative blot from 3 independent experiments).

 
3.2 EL mediates the binding and cell association of native and oxidized LDL
Since the uptake of LDL is dependent on its oxidation degree [23–25], we created mildly and extensively oxLDL by incubating native LDL with copper sulfate for different times. After subtracting non-specific binding/cell association (~30% of measurement), the average LDL binding for THP-1 macrophages was ~280 ng/mg cellular protein for native LDL, ~700 ng/mg cellular protein for mildly oxLDL, and ~1100 ng/mg cellular protein for extensively oxLDL (Fig. 2A, C, and E). Also, the amount of cell-associated LDL at 37 °C, most of which is internalized by cells [13], increased from ~1 µg/mg cellular protein for native LDL to ~1.5 µg/mg cellular protein and ~2.0 µg/mg cellular protein for mildly and extensively oxLDL, respectively (Fig. 2B, D, and F).


Figure 2
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  		Fig. 2 LDL binding and cell association in EL-suppressed THP-1 derived macrophages. THP-1 monocytes (5x105 cells/well) challenged with lentivirus containing either specific EL siRNA sequence (EL-LOF) or a scrambled siRNA sequence (Control) at a MOI of 20 for two days were transformed into macrophages by 100 nM PMA. LDL binding and cell association analyses were carried out as described in methods. Panel A and B: native LDL binding and cell association; Panel C and D: mildly oxLDL binding and cell association; Panel E and F: extensively oxLDL binding and cell association. (Statistical comparisons were made between cells transduced with a scrambled control and a specific EL target siRNA, n=8, **p<0.01, ***p<0.001].

 
The effects of either a loss-of-function or gain-of-function of EL on LDL binding and cell association were thereafter assessed. EL suppression (EL-LOF) was associated with significant reductions in the binding and cell association of native LDL of 53% and 33%, respectively (Fig. 2A and B). Similarly, EL suppression was also associated with significant decreases of 43% and 12% for the binding and cells association of mildly oxLDL (Fig. 2C and D). Furthermore, the binding of extensively oxLDL was significantly reduced by 36% after EL suppression but no significant differences were observed for the cell association of extensively oxLDL (Fig. 2E and F).

We then examined the role of increased EL expression on the same measured parameters. Overexpression of wild-type EL (EL-WT(GOF)) increased native LDL binding and cell association by 3.1- and 2.2-fold, respectively (Fig. 3A and B). Increased EL expression also markedly stimulated the binding and cell association of both mildly and extensively oxLDL, with 1.9- and 1.4-fold increase for mildly oxLDL and 1.5- and 1.5-fold increase for extensively oxLDL (Fig. 3C-F).


Figure 3
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  		Fig. 3 LDL binding and cell association in EL overexpressing THP-1 derived macrophages. THP-1 monocytes (5x105 cells) were challenged with lentivirus containing either wild-type EL cDNA (EL-WT(GOF)), S149A mutated EL (EL-S149A(GOF)), or no cDNA insert (Control) at a MOI of 20 for two days before treatment with 100 nM PMA to transform monocytes into macrophages. LDL binding and cell association analyses were carried out as described in methods. Panel A and B: native LDL binding and cell association; Panel C and D: mildly oxLDL binding and cell association; Panel E and F: extensively oxLDL binding and cell association. Statistical comparisons were made between cells transduced with no cDNA insert and the EL cDNA insert; n=4, **p<0.01, ***p<0.001].

 
3.3 The catalytic activity of EL does not play a critical role in LDL binding and cell association
To determine to what extent the catalytic activity of EL may have on the observed effects of LDL binding and cell association, we generated catalytically defective EL (EL-S149A) by mutating serine into alanine at position 149, and then performed LDL binding and cell association experiments again. Compared to wild-type EL (EL-WT), the catalytically inactive EL-S149A retained the same ability to mediate the binding of native, mildly and extensively oxidized LDL (Fig. 3A, C, and E). Also, catalytically inactive EL-S149A was still able to increase the cell association of LDL at all oxidative degrees, however, the extent to which it promoted LDL association was slightly less than those of wild-type EL, especially for native and mildly oxidized LDL (Fig. 3B, D, and F), suggesting that the catalytic activity of EL plays a minor but not critical role in EL-mediated LDL binding and cell association.

3.4 Heparan sulfate proteoglycans are required for EL-mediated LDL binding and cell association
Since several lipases including EL have been attributed non-catalytic or "bridging" function, we used heparinase I to eliminate this function of EL in wild-type EL-overexpressing THP-1 macrophages and subsequently analyzed LDL/oxLDL binding and cell association. Compared to untreated THP-1 macrophages, the heparinase I treatment reduced the basal binding of mildly and extensively oxLDL (49% and 60%, respectively), but its inhibition on basal binding of native LDL was not prominent (shaded bars, Fig. 4A, C, and E). The cell association of native LDL and extensively oxLDL was also significantly decreased in the presence of heparinase (49% and 15%, respectively). This inhibitory effect of heparinase I on basal LDL binding and cell association could be partially ascribed to the removal of the bridging function of basal EL and LPL, or other undetermined mechanisms [26,27].


Figure 4
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  		Fig. 4 Investigation of the role of HSPG on EL-mediated LDL binding and cell association in THP-1 macrophages. THP-1 monocytes were transduced by either no cDNA insert (Control) or wild-type EL-cDNA (EL-WT(GOF)) containing lentivirus and differentiated by PMA into mature macrophages as described in the methods, The binding and cell association of native and oxLDL were analyzed in the absence (no heparinase I) and the presence of 2.5 units/ml heparinase I (+Heparinase I). Panel A and B: native LDL binding and cell association; Panel C and D: mildly oxLDL binding and cell association; Panel E and F: extensively oxLDL binding and cell association (control, shaded bars; EL-mediated, non-overlapping clear bars. Statistical comparison between the presence and absence of heparinase I in control conditions: n=4; *p<0.05, **p<0.01, ***p<0.001; Statistical comparison between the presence and absence of heparinase I in EL-mediated (Control subtracted from EL-WT(GOF)) conditions: n=4; {dagger} p<0.05, {dagger} {dagger} {dagger} p<0.001).

 
As for the EL-mediated LDL binding, the presence of heparinase almost completely eliminated any enhanced effect of wild-type EL (EL-WT(GOF)) on LDL/oxLDL binding in THP-1 macrophages (Fig. 4A, C, and D). Similarly, significant decreases were also observed in EL-mediated cell association of all forms of applied LDL in the presence of heparinase I (Fig. 4B, D, and E). These results demonstrated that the preservation of the bridging function of EL is required for EL-mediated LDL binding and uptake.

3.5 The LDL receptor in-part facilitates the binding and cell association of native LDL mediated by EL expression
To investigate the potential role of specific cell surface receptors in the ability of EL to mediate LDL/oxLDL binding and cell association, we used reagents to block the receptor functions of the LDL receptor (LDLR), the LDLR-like protein (LRP) and CD36. The basal binding and cell association of native LDL were reduced by 49% and 84% by LDLR antibody (p<0.05), and 35% and 34% by LRP (p=0.07 and 0.06, shaded bars, Fig. 5A, B). As well, CD36 antibody blocked oxLDL binding and cell association by 32–58% (p<0.001, shaded bars, Fig. 5C–F).


Figure 5
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  		Fig. 5 Investigation of the role of LDLR, LRP and CD36 on EL-mediated LDL/oxLDL binding and cell association in THP-1 macrophages. THP-1 monocytes were transduced by either no cDNA insert (control) or wild-type ELcDNA (EL-WT(GOF)) containing lentivirus and differentiated by PMA into mature macrophages as described in the methods, The binding and cell association of native and oxLDL were analyzed in the presence (+) or absence (–) of specific blocking antibodies (LDLR-Ab and CD36-Ab) or RAP (LRP) to assess the role of these receptors. Panel A and B: native LDL binding and cell association; Panel C and D: mildly oxLDL binding and cell association; Panel E and F: extensively oxLDL binding and cell association. (control, shaded bars; EL-mediated, non-overlapping clear bars. Statistical comparison between the presence and absence of blocking agent in control conditions: n=4; *p<0.05, ***p<0.001; Statistical comparison between the presence and absence of blocking agent in EL-mediated (Control subtracted from EL-WT(GOF)) conditions: n=4; {dagger} p<0.05, {dagger} {dagger} p<0.01).

 
As far as EL-mediated LDL binding and cell association were concerned, antibodies specific for the LDLR moderately decreased the EL-mediated effects on native LDL binding and cell association by 24% and 54%, respectively (clear bars, Fig. 5A and B). However, in RAP pre-treated THP-1 macrophages, EL-mediated native LDL binding and cell association were not significantly affected (Fig. 5A and B). Although treatment of THP-1 macrophages with CD36 antibodies significantly decreased the binding and cell association of mildly and extensively oxLDL (p<0.001), no significant changes in EL-mediated binding and uptake of both mildly and extensively oxidized LDL were observed in the presence of CD36 monoclonal antibodies (Fig. 5C–F).

3.6 EL expression is associated with the lipid accumulation in THP-1 macrophages
After the suppression or overexpression of EL, the potential of THP-1 macrophage transformation into foam cells was evaluated by Oil Red O staining. As shown in Fig. 6, the extent of intracellular lipid accumulation is a function of the oxidation degree of LDL. EL suppression resulted in less lipid accumulation in all conditions when compared to control THP-1 macrophages (Fig. 6-1A–F). Furthermore, in THP-1 macrophages overexpressing wild-type EL (EL-WT), the level of intracellular lipid droplets after incubation with native, mildly and extensively oxidized LDL was significantly increased (Fig. 6-2B, 2E, and 2H). Consistent with LDL binding and cell association assays, the catalytic inactivation of EL (EL-S149A) did not compromise its ability to promote lipid accumulation in THP-1 macrophages with lipid levels comparable to wild-type EL (Fig. 6-2C, 2F and 2I). These observations show the evidence that EL expression in THP-1 macrophages can promote the lipid accumulation and lead to foam cell formation.


Figure 6
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  		Fig. 6 Oil Red O staining of macrophages. PMA differentiated macrophages which were either suppressed or overexpressing EL were incubated with 20 µg/ml LDL of different oxidation degrees for 24 h, and then stained with Oil Red O. Panel 1A, 1C, and 1E: control macrophages transduced with scramble-shRNA lentivirus (Control); Panel 1B, 1D, and 1F: EL-suppressed macrophages (EL-LOF). Panel 2A, 2D, and 2G: control macrophages transduced with blank lentivirus (Control); Panel 2B, 2E, and 2H: macrophages transduced with wild-type EL (EL-WT) by lentivirus; Panel 2C, 2F, and 2I: macrophages transduced with catalytically inactive EL (EL-S149A) by lentivirus.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
References
 
Although the influence of EL activity on the metabolism of circulating lipoproteins has been described, there is little knowledge about its tissue specific function in human macrophages. In the present study, we have used loss-of- and gain-of-function approaches to demonstrate that THP-1 macrophage-derived EL can mediate the binding and cell association of native LDL and oxLDL. A similar effect was also observed in transfected Chinese Hamster Ovary (CHO) cells, where the expression of recombinant EL markedly enhanced LDL binding by 3-fold [13]. With the prolonged incubation with LDL/oxLDL, EL overexpression significantly increased intracellular lipid accumulation in THP-1 macrophages, displaying an accelerating role in foam cell formation. This effect is parallel to the finding in COS cells where EL expression increased LDL internalization and degradation [13]. Therefore, macrophage-derived EL may promote foam cell formation and can be considered to be proatherogenic. Consistent with this concept is the report of the increased expression of EL in human atherosclerotic lesions and its colocalization with macrophages [9,28]. Similarly, this proposed role of EL would parallel the one described for a related lipase family member, lipoprotein lipase (LPL) [19–21].

We also observed that the catalytic activity of EL appeared to influence native LDL association but not binding which was observed when a catalytically inactive mutant form of EL was used. One possible explanation for this result is that the lipolytic modification of LDL by EL can change the LDL affinity for cells due to the reduction in size and surface molecule rearrangement following lipolysis. Therefore, this remodeled LDL particle may be more readily internalized by macrophages [29]. Consistent with our findings is the observation of reduced circulating apoB-containing lipoproteins in transgenic mice expressing catalytically active EL whereas catalytically inactive EL had the opposite effect [12], indicating the catalytic modification of lipoproteins could contribute to lipoprotein metabolism.

By contrast, the catalytic activity of EL in the present study assumes a less critical role in the cell association of oxidized LDL (especially extensively oxidized LDL) when compared to native LDL. Since these lipoproteins are recognized by different cell surface receptors in comparison to native LDL, the lipolytic modification of oxLDL by EL may not have comparable effects as observed with native LDL. Also, as indicated in previous reports [23–25], the oxidative modification of LDL markedly increases the LDL affinity for and metabolism by THP-1 macrophages when compared to native LDL. As a consequence, the affinity increase associated with lipolytic modification may be minimal in comparison to the greater change in the magnitude of binding and cell association associated with oxLDL catabolism.

In CHO cells, the catalytic activity of EL has been shown to be less critical than its non-catalytic or bridging function in the metabolism of apoB-containing lipoproteins [13]. The bridging function of EL is thought to be mediated by the conservation of clusters of positively charged amino acid residues in its C-terminus facilitating EL binding to cell surface HSPG [3]. To assess this aspect of EL function in LDL binding and cell association in EL over-expressing THP-1 macrophages, we treated cells with heparinase I to remove cell surface HSPGs. It was observed that the cell surface binding of EL is crucial for native LDL and oxLDL binding and cell association as heparinase treatment almost completely removed the enhancement of EL on both native and oxidized LDL binding and cell association. Like LPL [30–34], the presence of cell surface HSPGs is required for EL to mediate lipoprotein binding and subsequent internalization.

When we investigated the potential cooperative role of specific cell surface receptors, we identified that the LDLR is involved in the EL-dependent binding and cell association of native LDL. A similar relationship between the LDLR and LPL-mediated lipid uptake was reported indicating a 2-fold increased uptake of native LDL in LPL-treated aortic endothelial and HepG2 cells whereas virtually no increase in uptake was observed in LDLR-deficient cells [35,36]. Unlike LPL, which can interact with LRP to facilitate lipoprotein metabolism [37–40], LRP does not appear to be required for the EL-mediated lipoprotein removal. With regard to the EL-mediated uptake of oxLDL, blockage of CD36 did not have an appreciable effect. However, it is possible that additional scavenger receptors or receptor-independent mechanisms may contribute to this mechanism. For example, it has been shown that LPL can be internalized via HSPG and recycled [41], thus, a lipoprotein receptor-independent but heparitinase-sensitive metabolizing route has been proposed for LPL bound LDL [37,38,42].

In summary, THP-1 macrophage-derived EL expression enhances the binding and cell association of native and oxLDL as well as the lipid accumulation in human THP-1 macrophages, an effect dependent on the presence of cell surface HSPG. This data combined with our recent report that EL expression is associated with pro-inflammatory secretion in THP-1 macrophages [22] indicate that this lipase may have multiple effects on the atherogenic potential of the human macrophage.

Time for primary review 27 days


   Acknowledgements
 
This project was funded by grants from Canadian Institutes of Health Research (MOP74480), and the Heart and Stroke Foundation of British Columbia and Yukon. J.S. Hill is a Scholar of the Michael Smith Foundation of Health Research. G. Qiu is a recipient of the Heart and Stroke Foundation of Canada Doctoral Research Award.


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 

  1. Wong H., Schotz M.C. The lipase gene family. J Lipid Res (2002) 43:993–999.[Abstract/Free Full Text]
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